A quick, simple and unbiased method to quantify C2C12 myogenic differentiation

Introduction: C2C12 myoblasts undergo in vitro myogenesis to form protein‐rich multinucleated myotubes. Determining the fraction of total nuclei incorporated into myotubes is a commonly used method to quantify the extent of differentiation, but it is labor‐intensive and susceptible to operator bias. Methods: We have developed a simple method to quantify myotube formation using micrographs of Jenner–Giemsa‐stained C2C12 cultures. Because myotubes are darkly stained by Jenner–Giemsa dyes, the extent of myotube formation correlates with an increase in pixels attributed to the darkest tones. Thus, image histograms were obtained from photographs using ImageJ software, and the sum of the darkest tones was used as a measure of myotube density. Results: Measurements of myotube density mirrored those of fusion index during C2C12 differentiation and after treatment with prostaglandin D2, an inhibitor of C2C12 myogenesis. Conclusions: We propose this inexpensive, quick, and unbiased method to quantify C2C12 differentiation as a complement of the fusion index analysis. Muscle Nerve 44: 366–370, 2011